Equilibria and tyrosinase activity of a dinuclear and its analogous tetranuclear imidazolate-bridged copper(II) complexes

Inorganica Chimica Acta 321 (2001) 11–21

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Equilibria and tyrosinase activity of a dinuclear and its analogoustetranuclear imidazolate-bridged copper(II) complexes

Wendel Andrade Alves, Izilda A. Bagatin, Ana Maria Da Costa Ferreira *Departamento de Quımica Fundamental, Instituto de Quımica, Uni�ersidade de Sao Paulo, P.O. Box 26077, Sao Paulo 05513-970, SP, Brazil

Received 24 January 2001; accepted 17 May 2001

Abstract

A copper(II) complex, [Cu(apip)(imH)]2+, containing an imidazole ligand, in addition to a discrete tridentate imine (apip=2-[2-(2-pyridyl)ethylimino-1-ethyl]pyridine), was prepared. Both mono- and dinuclear species coexisted in aqueous solutionexhibiting a pH-dependent monomer�dimer interconversion and were monitored by different techniques. Similar data wereobtained with the complex [SECu(imH)]+, where SE=2-salicylidene-aminoethane. Moreover, these species were isolated asperchlorate salts and characterized by cyclic voltammetry and spectroscopic techniques (UV–Vis, IR, Raman, and EPR).Experiments by capillary electrophoresis, in addition to parallel EPR spectra at different pH values, permitted to estimate theequilibrium constant, corresponding to the equation: 2[LCu(imH)]2+ +OH− � [LCu(im)CuL]3+ +H2O+ imH, K= (8�3)×107 mol−1 dm3, in reasonable agreement to the value determined by spectrophotometric measurements, K= (0.12�0.01)×107

mol−1 dm3. A further tetranuclear species, [Cu4(apip)4im4]4+, was obtained by a pH-dependent self-assembly process, separatedfrom very alkaline solutions of the corresponding mononuclear complex. The tetranuclear and dinuclear species were observed tobehave as good functional models of the tyrosinase enzyme, catalyzing the aerobic oxidation of 2,6-di-tert-butylphenol, inmethanolic solution, and that of 3,4-dihydroxyphenylalanine (L-dopa) in aqueous solution, with a biphasic behavior in the rangeof pH 7–11. Applying the Michaelis–Menten approach, the kinetic parameters determined indicated that the tetranuclear speciesis a better catalyst than the dinuclear ones, exhibiting a higher rate constant, k3, as well as a higher KM value. The hydrophobiccavity in the polynuclear complex seems to facilitate its interaction with the phenol substrate, since the structural parametersdetermined for both the complexes, di- and tetranuclear species were very similar. © 2001 Elsevier Science B.V. All rightsreserved.

Keywords: Copper complexes; Imidazolate-bridged complexes; Tyrosinase activity

1. Introduction

The imidazole ring is an ubiquitous essential metalbinding site in different metalloproteins [1], occurringas histidine side chains in blue copper proteins, hemo-cyanin, tyrosinase, multicopper oxidases, and cy-tochrome c oxidase. Additionally, this group can alsoact as a bridging ligand between different metal centers,as in the Cu2, Zn2 superoxide dismutase [2]. Therefore,a considerable number of imidazole metal complexes,specially those involving imidazolate-bridged dicoppercenters, have been prepared and characterized, in order

to understand the peculiar properties of these naturalsystems better. Most of these model compounds werestabilized by incorporating the dicopper-imidazolatemoiety into macrocyclic ligands [3–6]; others, by usingpolycoordinating imidazole derivatives [7–9]. However,some were prepared successfully as ternary species withsimple ligands, such as polyamines, diimines, or dipep-tides [10–12]. The main goal in many of these studieswas to mimic and explore structural characteristics ofactive centers in copper proteins, particularly throughX-ray structure determination, magnetic properties, andEPR spectra. Also, some systems were investigated inorder to obtain stable dioxygen species [LCuO2CuL], inanalogy to oxyhemocyanin [13]. In contrast, the stabil-ity and the catalytic activity of these complexes inoxidation reactions of suitable substrates were less in-

* Corresponding author. Tel.: +55-11-3818-2151; fax: +55-11-3815-5579.

E-mail address: [email protected] (A.M. Da Costa Ferreira).

0020-1693/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.

PII: S 0 0 2 0 -1693 (01 )00490 -X

W.A. Al�es et al. / Inorganica Chimica Acta 321 (2001) 11–2112

vestigated, in comparison to analogous pyridyl or pyra-zolyl derivatives [14].

Previous studies on diimine copper complexes havebeen developed in our lab, focusing mainly their SODactivity and their reactivity toward hydrogen peroxide[15]. In this work, we described a study on the stabilityand the catalytic activity toward the aerobic oxidationof phenolic substrates of some copper(II) complexescontaining an imidazole ligand, in addition to a discretetridentate imine, shown in Fig. 1. The compoundsstudied, with general formula [CuL(imH)]n+, exhibitedin aqueous solution a dimerization equilibrium,strongly dependent on the pH of the solution, formingthe corresponding imidazolate-bridged species,[LCu(im)CuL]. Therefore, both the mononuclear andthe corresponding dinuclear species were isolated andcharacterized by elemental analysis, UV–Vis, IR, Ra-man and EPR spectroscopies, and magnetic susceptibil-ity measurements. The dinuclear complexes were theninvestigated as potential catalysts in the oxidation of2,6-di-tert-butylphenol, in methanol solution, and of3,4-dihydroxyphenylalanine (L-dopa), in aqueous solu-tion, acting as functional models of the tyrosinaseenzyme. Further, a tetranuclear species, [Cu4L4(im)4],was isolated with one of the ligands (L=2-[2-(2-pyridyl)ethylimino-1-ethyl]pyridine), in very alkaline so-lution, and also had its reactivity studied, incomparison to the dinuclear complexes.

2. Experimental

2.1. Materials

All reagents were of analytical grade, purchased fromdifferent sources and used without further purification.The following abbreviations were used: imH, imidazole;im, imidazolate anion; SE, 2-salicylidene-aminoethane,ligand derived from salicylaldehyde and 1,2-di-aminoethane; apip, 2-[2-(2-pyridyl)ethylimino-1-ethyl]-pyridine, ligand derived from 2(2-aminoethyl)pyridineand 2-acetylpyridine.

2.2. Syntheses of the copper complexes

Warning: Perchlorate salts of metal complexes withorganic ligands are potentially explosive and should behandled very carefully, only in small amounts.

2.2.1. [SECu(imH)]ClO4 and [SECu(im)CuSE]ClO4

The complex [SECu(imH)]ClO4 (1) and the corre-sponding dinuclear species [SECu(im)CuSE]ClO4 (2)were prepared in small amounts (2 or 4 mmol), accord-ing to previously described procedure [12]. Yields: 84and 94%, respectively. For compound 1. Anal. Found:C, 37.58; H, 3.85; Cu, 16.13; N, 13.92. Calc. forC12H15N4OCu(ClO4): C, 36.56; H, 3.83; Cu, 16.10; N,14.21%. IR: �(C�N), 1480s, 1642s; �(C�N), 1095s;�(C�O), 1300s; �(N�H), 3 319w, 3268w. For compound

Fig. 1. Schematic structure of the complexes studied.

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2. Anal. Found: C, 39.69; H, 3.73; Cu, 20.56; N, 13.63.Calc. for C21H25N6O2Cu2(ClO4): C, 40.68; H, 4.06; Cu,20.50; N, 13.55%. IR: �(C�N), 1480s, 1642s; �(C�N),1095s; �(C�O), 1300s; �(N�H), 3319w, 3272w.

2.2.2. [Cu(apip)imH](ClO4)2

The compound [Cu(apip)imH](ClO4)2 (3) was ob-tained as deep blue crystals, by a similar procedurereported for related complexes [10]. Briefly, themononuclear complex was obtained by dropping 1.2 ml(10 mmol) of 2-(2-aminoethyl)pyridine into 1.1 ml (10mmol) of 2-acetylpyridine dissolved in 50 ml ofmethanol, under stirring. After a few minutes, 10 mmol(3.7 g) of copper perchlorate hexahydrate dissolved in10 ml of water were added, followed by 1.4 g (20 mmol)of imidazole. The resulting solution was maintainedunder continuous stirring, at room temperature (r.t.),for 8 h, and after that, in ice bath for 20 min. Deep bluecrystals were collected by filtration, washed with cooledmethanol, diethyl ether and finally dried in vacuum.The obtained yield was 72%. Anal. Found: C, 36.82; H,3.45; Cu, 11.85; N, 12.50. Calc. for C17H19N5Cu(ClO4)2:C, 36.73; H, 3.44; Cu, 11.43; N, 12.60%. IR: �(OH),3490s; �(C�N), 1490s, 1636s; �(C�N), 1090s; �(N�H),3425w.

2.2.3. [Cu2(apip)2im](ClO4)3 ·H2OThe dinuclear species, [Cu2(apip)2im](ClO4)3·H2O (4),

was prepared similarly by dissolving 0.36 ml (3.0 mmol)of 2(2-aminoethyl)pyridine in 10 ml of methanol anddropping this solution to 0.34 ml of 2-acetylpyridine,dissolved in 10 ml of methanol, followed by the addi-tion of 1.18 g (3.0 mmol) of copper(II) perchloratehexahydrate previously dissolved in 10 ml of deionizedwater and 0.102 g (1.5 mmol) of imidazole. The pH ofthis solution was then adjusted to 9.40, by adding 1.5ml of NaOH solution (1.0 mol dm−3). The final darkblue solution was then stirred for approximately 8 h, atr.t., when a blue precipitate was formed. The solutionwas cooled in ice bath and the crystals obtained werefiltered, washed with cold ethanol and diethyl ether anddried in vacuum. Yield: 92%. Anal. Found: C, 38.28; H,3.75; Cu, 12.76; N, 11.37. Calc. for C31H35N8OCu2-(ClO4)3: C, 38.74; H, 3.67; Cu, 13.21; N, 11.65%. IR:�(OH), 3443s; �(C�N), 1485m, 1643m.

2.2.4. [Cu4(apip)4(im)4](ClO4)4 ·2H2OA tetranuclear species, [Cu4(apip)4(im)4](ClO4)4·2H2O

(5), was also obtained, by adding 0.35 ml (2.54 mmol)of triethylamine to a solution of the correspondingmonomer complex, [Cu(apip)imH](ClO4)2 (3), 0.90 g(1.5 mmol) dissolved in 15 ml of ethanol. After stirringthis solution for 30 min, the formation of a precipitatewas observed. By cooling the solution in ice bath, lightblue crystals were formed and collected by filtration,washed with ethanol and diethyl ether and finally dried

in vacuum. Yield: 87%. Anal. Found: C, 41.43; H, 3.87;Cu, 13.45; N, 13.42%. Calc. for C68H76N20O2Cu4-(ClO4)4: C, 43.97; H, 4.12; Cu, 13.68; N, 15.08%. IR:�(OH), 3441s; �(C�N), 1490s, 1636m; �(C�N), 1090s.

2.2.5. [Cu(apip)OH]ClO4

For comparative purposes, this complex was alsoprepared, in very similar conditions of the compound[Cu(apip)imH](ClO4)2 (6), without the addition of imi-dazole. The yield obtained was 74%. Anal. Found: C,41.13; H, 3.95; Cu, 15.47; N, 11.43. Calc. forC14H16N3OCu(ClO4): C, 41.48; H, 3.99; Cu, 15.68; N,10.37%. IR: �(OH), 3443s; �(C�N), 1492s, 1639s;�(C�N), 1092s.

2.3. Physical measurements

Elemental analyses were performed at the Nucleo deInstrumentacao Analıtica of our Institution, using aPerkin–Elmer 2400 CHN Elemental Analyser. Copperanalysis was carried out by atomic emission spectrome-try, in an ICP-AES Spectroflame spectrometer, moni-tored at 327, 396 nm, within a detection limit of 4.5�g dm−3. Electronic spectra were registered in a Beck-man DU-70 spectrophotometer, or an Olis modernized-Aminco DW 2000 instrument, with thermostated cellcompartment. EPR spectra were recorded in a BrukerEMX instrument, operating at X-band frequency, usingstandard Wilmad quartz tubes, at 77 K. DPPH (�,��-diphenyl-�-picrylhydrazyl) was used as frequency cali-brant (g=2.0036) for powder samples and[Cu(edta)]2+ solutions (1.00 mmol dm−3) as standardfor frozen solutions. Infrared spectra of the complexesobtained were recorded in a BOMEM 3.0 instrument,in the range 4000–200 cm−1, using KBr pellets. Ramanspectra were registered in a Renishaw Ramascope 3000spectrometer, equipped with a CCD detector and anOlympus BTH2 microscope (80-fold enhancing), usingrotating Teflon cell, and acquisition time of 5 s, withdifferent accumulation. Excitation of sample was set upat 632.8 nm, with a He–Ne laser, model 127, fromSpectra Physics.

Capillary electrophoresis experiments were per-formed in a P/ACE 5510 Beckman instrument,equipped with a diode array detector and a SystemGoldTM software. A silica capillary (75 �m×375 �m×50 cm) kept at 25 °C was used. Samples were intro-duced at 0.5 psi and 20 kV, in phosphate buffer (5.0mmol dm−3) of different pH values, monitored a 214nm. Magnetic susceptibilities were determined in aCAHN Faraday balance, model DTL 7500, using mer-cury tetra(thiocyanato)cobaltate(II), [HgCo(SCN)4], asstandard (�=16.44×10−6 CGS/G units, at 20 °C and�= +10° [16]. Effective magnetic momentum valueswere calculated by the equation: �eff=2.828(�MT)1/2,

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Table 1Spectral properties of imine copper(II) complexes containing an imidazole ligand

Compound �max (nm) (�, M−1 cm−1)�max (nm) (�, 103 M−1 cm−1)

Intraligand transitionsn��, or n��* transitions d–d band���*

[SECu(imH)]ClO4 (1) 196 (17.3), 216 (21.2) 235 (21.2), 263 (11.1) 350 (4.4) 613 (100)350 (6.6)[SECu(im)CuSE]ClO4 (2) 605 (133)196 (28.9), 216 (31.3), 235 (33.5), 263 (17.0)

203 (29.8), 254 (8.6), 282 (7.8) 636 (68)[Cu(apip)imH](ClO4)2 (3)[Cu2(apip)2im](ClO4)3·H2O (4) 630 (137)200 (59.6), 254 (17.5), 282 (15.5)

200 (59.7), 254 (17.4), 282 (15.2) 624 (158)[Cu4(apip)4(im)4](ClO4)4·2H2O (5)[Cu(apip)OH](ClO4) (6) 202 (18.2), 264 (5.5), 284 (3.8) 650 (56)

Spectra in aqueous solutions.

after the appropriate Pascal constant corrections for thecorresponding diamagnetic contributions.

The pH of the solutions was monitored in a DigimedDMPH-2 instrument, coupled to a combined pH elec-trode, from Ingold or Radiometer. Appropriate buffersolutions were used to calibrate the instrument. Con-ductivity experiments with the complexes studied (in 1mmol dm−3 aqueous solution) were carried out in aDigimed DM-31 instrument, using a 10.0 mmol dm−3

KCl solution as standard (specific conductivity=1412.0 �S cm−1, at 25 °C) [17].

Cyclic voltammetric data were collected under argonatmosphere, using a Princetom Applied Research Corp.(PARC) instrument, consisting on a 175 universal pro-grammer, a 173 potentiostat and a XY recorder. Thecomplexes (5 mmol dm−3) were dissolved in water con-taining KCl (0.25 mol dm−3). Glassy carbon was usedas working electrode, Pt wire and Ag/AgCl were usedas auxiliary and reference electrode, respectively. Poten-tials were referenced to SHE by adding 0.222 V. Spec-troelectrochemical measurements were conducted in aHewlett Packard 8452 A diode array spectrophotome-ter combined with a PARC potentiostat, model 173, inaqueous solution (1 mmol dm−3) containing KCl (0.25mol dm−3). A quartz spectroelectrochemical cell with0.025 cm optical length and a gold minigrid as workingelectrode was used.

2.4. Kinetic studies

The catalyzed oxidation of 2,6-di-tert-butylphenolwas performed under pseudo-first order conditions, in astandard quartz cell with 10 mm optical length and 3.00ml volume, at 25.0�0.5 °C, in methanol solution,following the formation of the corresponding diquinoneor diphenoquinone at 418 nm (�=5.48×104

mol−1 l cm−1). Experimental curves of increase in ab-sorbance as a function of time were analyzed by initialrate method. Deviations in values of rate constantswere �5% as indicated, estimated by repeated experi-

ments. Analogous experiments with 3,4-dihydrox-yphenylalanine (L-dopa) were carried out at30.0�0.5 °C, in aqueous solutions, monitoring thequinone formed at 475 nm (�=3.60×103

mol−1 l cm−1). For these experiments, the standardconcentrations used were [L-dopa]=6.67 mmol dm−3

and [catalyst]=0.145 mmol dm−3, at pH 7.3 or 11.0.

3. Results and discussion

The mononuclear copper(II) complexes [SE-Cu(imH)]ClO4 (1) and [Cu(apip)imH](ClO4)2 (3), con-taining an imidazole ligand in addition to a tridentateimine, were prepared by standard methods in the litera-ture, with minor suitable modifications [10,12]. Thesecompounds showed a rapid dimerization equilibrium inaqueous solution, highly dependent on the pH, leadingto the corresponding dinuclear species, [SE-Cu(im)CuSE]ClO4 (2) and [Cu2(apip)2im](ClO4)3·H2O(4), respectively, with an imidazolate-bridged ligand,which were also isolated and characterized by UV–Vis,IR and EPR spectroscopy. In addition, a tetranuclearspecies, [Cu4(apip)4(im)4](ClO4)4·2H2O (5), was isolatedfrom alkaline solutions (pH�9) of compound 3, asdescribed in Section 2. Schematic structures of thecomplexes prepared are shown in Fig. 1.

3.1. Characterization of the complexes prepared

The UV–Vis spectra of the complexes studied inaqueous and 50% DMSO solutions showed intenseabsorption bands in the range 190–340 nm, usuallyattributed to n�� and ���* transitions in the lig-ands, and a characteristic copper d–d band around600–640 nm (see Table 1). Spectra of the mononuclearand the corresponding dinuclear complexes were verysimilar, although the d–d band usually occurred atlower wavelength in the dinuclear species. For the seriesmono-, di- and tetranuclear species, obtained with L=

W.A. Al�es et al. / Inorganica Chimica Acta 321 (2001) 11–21 15

apip, the observed �max shifted from 636 to 630 and 624nm, respectively. Intraligand ���* transitions of thesalicylaldimino chromophore [18] can explain the bandobserved around 350 nm for the [SECu(imH)]+ and[SECu(im)CuSE]+ complexes.

The registered IR spectra in KBr pellets showed theexpected characteristic bands of diimine compounds, asindicated in Section 2. The stretching frequencies forC�H bonds in aliphatic CH, CH2 and CH3 groups, oraromatic C�H bonds were observed around 3070–2921cm−1; bands attributed to aliphatic or aromatic CHbending, in the region 993–738 cm−1, were also ver-ified. The characteristic C�N bond stretching was foundat 1672–1600 cm−1, followed by aromatic C�C stretch-ing at 1565–1526 cm−1. Bands commonly ascribed toaliphatic Cu�N bonds appeared at 460–440 cm−1 andthe aromatic ones at 280–208 cm−1. All the complexesprepared showed additionally bands at 1090 and 630cm−1, characteristic of non-coordinated perchlorateions.

Usually, bands at 3450–3200 cm−1 have been as-signed to �N�H in IR spectra, but as the strong absorp-tion of �O�H also occurs in this region, ambiguousassignment are frequent [19]. In our mononuclear com-plexes, such bands occurred at 3319 and 3268 cm−1 for1 and 3425 cm−1 for 3, assigned to the N�H bond inthe imidazole ring.

The main difference among mono- and di- or te-tranuclear species, consisting on the N�H bond in theimidazole ring, was then monitored by Raman spec-troscopy (shown in Fig. 2(A)). Two characteristic bandswere verified in the Raman spectra of the mononuclearcomplex [Cu(apip)imH](ClO4)2 (3), at 235 and 265cm−1, assigned to the stretching frequency of Cu�NimH

and Cu�Npy bonds, respectively. On the other hand, inthe spectra of [Cu2(apip)2im](ClO4)3·H2O (4) and[Cu4(apip)4(im)4](ClO4)4·2H2O (5), two bands occurredin the region of the Cu�Nim stretching, at 195 and 215cm−1 and were attributed to different types of Cu�Nbonds: the lower one to the Cu�N� and the higher oneto the Cu�N� [20].

Differences in the spectra of these compounds at highfrequency region, around 2700–3200 cm−1, were alsoverified. For compound 3 bands at 2914, 2950, 3000,3091, 3152, and 3175 cm−1 were observed, referred toN�H, CH2, and CH3 bond stretching (see Fig. 2(B)).Particularly, the bands at 3152 and 3175 cm−1 can beascribed to the characteristic �N�H of the imidazole ring,since these bands do not occur in the spectra of theanalogous di- or tetranuclear species, compounds 4 and5, and also in the spectrum of a similar compound,[Cu(apip)OH]ClO4 (6), not containing the imidazolegroup.

In Table 2, the magnetic properties of these com-plexes are illustrated, showing that in the polynuclearspecies some antiferromagnetic interaction occur, inspite of the large distance between the copper centers,as indicated by smaller values of the effective magneticmomentum per copper (�eff), in comparison to thecorresponding mononuclear complex. For instance, inthe series with L=apip, the mononuclear complex hasa �eff=2.10 �B, while the analogous dinuclear andtetranuclear species exhibited values of 1.88 and 1.59 �B

per copper, respectively, smaller than the expected spin-only value. Similar values were observed for the [SE-Cu(imH)]+ and [SECu(im)CuSE]+ complexes, with�eff=1.78 and 1.35 �B, respectively. Although small,the differences observed are significant, as compared tosimilar compounds previously described in the litera-ture [21,22].

In order to confirm the ionic nature of the speciesprepared, conductivity measurements were also per-formed. Molar conductance of these complexes, in 1.00mmol dm−3 aqueous or DMF solutions, indicated val-ues consistent with the proposed formula, as shown inTable 2. Both compounds 1 and 2 exhibited �M values

Fig. 2. Raman spectra of the studied copper complexes in solid state:(A) at the low wavenumber range (100–400 cm−1) and (B) at thehigh wavenumber range (2000–4000 cm−1).

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Table 2Magnetic properties, molar conductance and EPR parameters of the copper complexes studied

�Ma (S cm2 mol−1 at 298 K) EPR parameters bCompound �eff, (�B at 295 K)

g� g� A� (10−4cm−1) g�/A� (cm)

109.2 2.056 2.229[Cu(imH)SE]ClO4 (1) 1791.78 124138.1 2.059 2.2281.35 177[SECu(im)CuSE]ClO4 (2) 126

2.10[Cu(apip)imH](ClO4)2 (3) 238.0 2.053 2.248 164 1371.88[Cu2(apip)2im](ClO4)3·H2O (4) 339.0 2.056 2.245 166 135

558.0 2.060 2.2401.59 166[Cu4(apip)4(im)4](ClO4)4·C2H5OH (5) 1351.90[Cu(apip)OH]ClO4 (6) 129.4 2.057 2.288 116 197

a In aqueous solution.b In frozen 50% DMSO aqueous solution at 77 K.

in the range 100–130 S cm2 mol−1, behaving as 1:1electrolytes, while complex 3 indicated a 238S cm2 mol−1 value, expected for a 1:2 electrolyte, inaqueous solution. The obtained �M value for dinuclearcomplex 4, 339 S cm2 mol−1, corroborated their behav-ior as tri-cationic species. Compound 6 corresponds toa 1:1 electrolyte, with �M=129 S cm2 mol−1. Finally,for tetranuclear complex 5 the molar conductance val-ues obtained, 558 or 139 S cm2 mol−1 per copper,indicated a 1:4 electrolyte. Apparently, there is noevidence for oligomeric or infinite zig-zag chains [23] inany of these compounds. Our results are in close agree-ment with recently described data for similar cyclic-te-trameric copper(II) species, obtained by self-assemblyprocesses [22]. These complexes, containing imidazole-derived moieties, exhibited electrical conductivity of 99S cm2 mol−1 per copper, in water, and effective mag-netic moments of 1.57 �B [24].

With the aim at comparing the geometric environ-ment around copper ions in those complexes, spectro-scopic studies by EPR were then carried out. Spectra infrozen 50% aqueous DMSO solutions, at 77 K, areshown in Fig. 3, illustrating both the signals at g�2,around 3100 G, and also at g�4, around 1500 G. Thecorresponding parameters determined are shown inTable 2.

Some remarkable differences in these spectra wereobserved, although equilibria can be present in thesesolutions. First, a superhyperfine structure multipletowing to 14N atoms around the copper ion was ob-served in the g� signal in the series of complexes withthe ligand apip, as shown more appropriately in Fig. 4.This multiplet showed nine lines (better resolved inamplified spectra, not shown), with AN�=14.7 G, inthe mono-, di- and tetranuclear species, as expected forat least four 14N atoms strongly coordinated to copperin an approximately tetragonal ligand field [25]. On theother hand, this multiplet was not observed in the[Cu(apip)OH]+ complex, not containing the imidazolegroup, and exhibited five lines in the correspondingmono- and dinuclear species, [SECu(imH)]+ and [SE-

Cu(im)CuSE]+, with only two 14N atoms coordinated.Moreover, the spectra exhibited a characteristic signalaround 1500 G, attributed to �M= �2 transitions incenters with Cu···Cu magnetic exchange interaction

Fig. 3. EPR spectra of the copper complexes studied (�5 mM), infrozen 50% DMSO solutions, at the g�2 and g�4 regions (�100X). (A) [Cu(apip)imH], gain=3.99×103, 7.10×105;[Cu2(apip)2im], gain=1.12×104, 1.42×105; [Cu4(apip)4(im)4],gain=7.10×103, 3.17×105. (B) [SECu(imH)], gain=1.42×104,1.42×106; [SECu(im)CuSE], gain=2.24×103, 3.56×105.[Cu(apip)OH]=1.12×104, 3.17×105.

W.A. Al�es et al. / Inorganica Chimica Acta 321 (2001) 11–21 17

Fig. 4. EPR spectra of the copper complexes studied, in frozenmethanol–water (4:1, v/v) solutions, at 77 K. Gain=3.17×104, for[Cu(apip)OH]+ and [Cu(apip)imH]2+ complexes, and 4.48×104 for[Cu2(apip)2im]3+, [Cu4(apip)4(im)4]4+ complexes.

3.2. Equilibria studies

An equilibrium strongly dependent on the pH wasobserved in aqueous solutions involving complexes 3and 4, expressed by:

2[LCu(imH)]2+ +OH− � [LCu(im)CuL]3+ +H2O

+ imH (1)

with K= [D][imH]/[M]2[OH−], where D=dinuclearand M=mononuclear species.

Spectral changes were observed in the d–d band withan increasing absorbance, and a shift to lower wave-length as the pH increased, expected from the forma-tion of the dinuclear species. Similar results were alsoobtained for complexes 1 and 2, although these specieswere verified to be more unstable than the former ones.

By using capillary electrophoresis, it was possible toseparate the mono- and the dinuclear species, com-plexes 3 and 4, respectively, as shown in the electro-pherograms in Fig. 5. In these experiments phosphatesolutions at different pH values were used as eluent. Asa tri-cationic species, the dinuclear complex was elutedfirst, showing an increasing elution rate with increasingpH. Further, by confronting these data with the EPRspectra at different pH (shown in Fig. 6), it seems thatat pH�12 the imidazole ligand is probably replaced bya hydroxo ligand, forming compound 6, according tothe equilibrium:

[LCu(im)CuL]3+ +OH−+H2O � 2[LCu(OH)]+

+ imH (2)

As indicated in the electropherograms, the peak areain Fig. 5, corresponding to pH 12, attributed to thefinal hydroxo complex, corresponds to the sum of the

Fig. 5. Electropherograms of [Cu(apip)imH]2+ solutions at differentpH values. Species were monitored at 214 nm, at 25.0�0.5 °C, usingphosphate solutions as eluent, over the pH range 5–12.

Fig. 6. EPR spectra of the [Cu(apip)imH]+ complex in frozen 50%DMSO solutions (77 K), at different pH values: 6.95 (gain=5.02×105), 8.30 (2.83×105), 8.93 (7.10×105), 9.72 (5.64×105), 10.35(4.48×105), 11.55 (6.32×105).

[26]. This signal, observed in the spectra of the dinu-clear and tetranuclear species of the complexes studied(vide Fig. 3), showed a hyperfine structure with sevenlines, and a hyperfine parameter of 75.5 G, comparableto reported values for similar complexes [4,5,21,25,27].

Based on these data, a square-pyramidal coordina-tion geometry is expected in the case of the tetranuclearcomplex 5, in analogy to a similar cyclic complex whoseX-ray analysis has been published [24]. For the mono-and dinuclear species, a distorted tetragonal copper sitecould be inferred, by comparing the empirical ratiog�/A�, frequently used to evaluate tetrahedral distortionin structural features of copper SOD mimetic com-pounds [28]. This distortion was observed to be muchstronger in 6, not containing the imidazole group.

W.A. Al�es et al. / Inorganica Chimica Acta 321 (2001) 11–2118

Fig. 7. pH-dependent electronic spectra of the [Cu(apip)imH]+ com-plex (3.00 mmol l−1) in 50% DMSO aqueous solution, upon additionof 0.1 mol l−1 NaOH solution. From bottom to top, pH 6.44, 7.35,8.00, 8.80, 9.25, 9.41, 9.72, 9.97, 10.39, 10.58, 10.90, 11.17, and 11.67.

sponding K constant, based on a general method forspectrophotometric data [29]. From equilibrium in Eq.(1), 2 log [D]/[M]= log K−14+pH; therefore a plot oflog [D]2/[M]2 versus pH should be a straight line withslope 1 and intercept log K−14, since (A0−A+)/(A+ −A�)= [2c2]/[c1], where A0=�1ctot=�1(c1+2c2),A�=�2c2�, A+ =�1c1+�2c2, and c1 and c2 are theconcentration of the mono- and dinuclear complexes,respectively.

At pH 6.44 the mononuclear species is predominantand the dinuclear species can be neglected, based onEPR data. On the other hand, the spectrum at pH 9.72corresponds primarily to the dinuclear complex. Byconsidering the absorptivity values for the d–d banddetermined for each species (58 and 159 M−1 cm−1, forthe mono- and dinuclear species, respectively) and thatimidazole do not contribute to absorbance at this spec-tral region, the calculated equilibrium constant at 653nm, in the range of pH 6.44–9.72, was (0.12�0.01)×107 mol−1 dm3. This value is in reasonable agreementwith the value previously estimated from the capillaryelectrophoresis data, considering the different accura-cies of these methods.

3.3. Electrochemical measurements

Cyclic voltammograms in aqueous solution indicateda reversible behavior of the mononuclear[Cu(apip)imH]2+ species in the range of pH 5.35–8.4,which presented a well-defined couple wave at E1/2= −0.05 V versus SHE, in a one-electron process corre-sponding to the redox pair CuIIL/CuIL (shown in Fig.8). When the voltammograms were performed at pH�5.0, a small stripping peak appears near 0.0 V, assignedto the Cu(0) to Cu(II) oxidation, confirming the lowstability of the reduced compound in more acidic media[30]. Spectroelectrochemical data were then obtained inaqueous solution, in the range 0.22 to −0.28 V versusSHE, at pH 5.35. By scanning to negative potential, itwas possible to see a strong decrease of the ���*ligand bands at 258 and 283 nm, with the simultaneousincreasing of a broad band at 470 nm, assigned as a CTCu(I)� imine band, also observed in some macroa-cyclic copper(I) complexes [31].

The dinuclear complex, on the contrary, exhibited atpH 9.39 an irreversible wave, at EPc= −0.07 V versusSHE, assigned to a Cu(II)/Cu(I) reduction, with associ-ated peaks at EPa=0.10 and 0.30 V versus SHE (videFig. 9). After the reduction, the dissociation of theimidazole bridged ligand can occur, giving probably the[Cu(apip)OH]+ species, according to similar studiesreported in the literature [32]. Repeated scanning overthe range +0.20 to −0.40 V caused a little decay inthe intensity of these peaks, demonstrating a decompo-sition process on the electrode surface. The peak at 0.30V can be attributed to the Cu(0) to Cu(II) oxidation,

mono- and dinuclear signals in Fig. 5 in the range ofpH�8, while the areas for the peaks attributed to thedinuclear species, observed at pH 8–10, are approxi-mately half this value. By determining the correspond-ing peak areas, at the different pH values, a value forthe equilibrium constant in Eq. (1), involving themono- and dinuclear species, K= (8�3)×107

mol−1 dm3 was estimated. Similar equilibria have beensuggested for the 1,1,7,7-tetramethyldiethylenetriamine-copper(II) complex, with predominance of the corre-sponding hydroxo species at pH�9; however, in thiscase the equilibrium constant has not been calculated[21].

In our case, a further equilibrium step could beconsidered, in the presence of excess imidazole, leadingto the previously isolated and characterized tetranu-clear species:

2[LCu(im)CuL]3+ +2imH+2OH− � [Cu4L4(im)4]4+

+2H2O (3)

Variations in the EPR spectra of complex 3 withincreasing pH were also used to monitor these equi-libria. In Fig. 6, spectra at different pH values, in therange 7–11, are displayed. These data corroboratedand complemented the electrophoresis results. The sig-nal at g�4, attributed to �MS= �2 transitions, dueto magnetic interaction between two copper centers[26], unequivocally indicated the formation of the dinu-clear, or the tetranuclear species at pH 8–11 and thefinal replacement of the imidazole group by the hy-droxo ligand, at even higher pH.

Variations in the electronic spectra of complex 3 in50% DMSO aqueous solution, at different pH values,shown in Fig. 7, were also used to estimate the corre-

W.A. Al�es et al. / Inorganica Chimica Acta 321 (2001) 11–21 19

which has been already observed for other coppercomplexes [33]. Therefore, these data point to a verystabilized mononuclear complex in both oxidationstate, while for the dinuclear species, predominant atpH 9.39, the electron transfer seems to involve a drasticstructural change, avoiding the sequential one-electron-reduction steps observed in very stable macrocycliccopper(II) complexes [34].

3.4. Phenol and catechol oxidase acti�ity

The catalytic activity of the complexes prepared inthe oxidation of phenolic substrates was then verified.Experimental curves in Fig. 10 showed that two of

Fig. 9. Cyclic voltammogram of the dinuclear [Cu2(apip)2im]3+

complex in aqueous solution (pH 9.39), at 20 mV s−1 scan rate.

Fig. 8. Cyclic voltammograms and UV–Vis spectroelectrochemicalbehavior of the mononuclear [Cu(apip)imH]2+ complex in aqueoussolution (pH 5.35).

them efficiently catalyze the oxidation of 2,6-di-tert-butylphenol, a hindered phenol that favored the forma-tion of the corresponding quinone or diphenoquinone.The tetranuclear [Cu4(apip)4(im)4]4+ compound wasthe most catalytically active species, while the [SE-Cu(im)CuSE]+ complex was inactive. Even themononuclear [Cu(apip)imH]2+ species exhibited someactivity, attributed to the dinuclear species fractionpresent in solution. They also catalyzed the oxidationof L-dopa, in aqueous solution in the range of pH7–11, forming the corresponding quinone, monitoredat 475 nm, as a biphasic process.

The rate law determined for the phenol oxidationindicated a first order dependence on the catalyst, anda first order dependence followed by a saturation effecton the substrate concentration, for both the complexes,as shown in Fig. 11. However, the dinuclear complexexhibited a higher order for [catalyst]�4×10−5

mol l−1, probably due to the formation of the tetranu-clear species, favored at higher concentrations. Theseresults agree well with the catalytic cycle usually pro-posed for the tyrosinase activity of dinuclear coppercompounds [35], based on the initial reduction of themetal centers by the phenolic substrate, followed by thecoordination of the molecular oxygen to the copperions. By applying the traditional Michaelis–Mentenapproach, values for the k3 and KM kinetic parameterswere obtained from the corresponding 1/Vi versus1/[phenol] curves: k3= (1.20�0.05) and (2.40�0.08)×10−2 min−1 and KM= (1.80�0.08) and (5.2�0.2) mmol dm−3, respectively, for the dinuclear[Cu2(apip)2im]3+ and tetranuclear [Cu4(apip)4(im)4]4+

W.A. Al�es et al. / Inorganica Chimica Acta 321 (2001) 11–2120

complexes. In the Michaelis–Menten scheme, k3 is themaximal velocity at saturating concentrations of thesubstrate and KM is the dissociation constant of thesubstrate S from the catalyst–substrate complex [36]:

Catalyst+Substrate �k1

k 2

[Catalyst···Substrate]�k3

Product

+Catalyst

Our data can be compared to other catalytic species,such as a dinuclear copper(II) complex derived from atriamino pentabenzimidazole ligand, described recentlyin the literature, with kcat=k3/[catalyst]= (1.40�0.06)s−1 and KM= (1.5�0.2) mmol dm−3, for the oxida-tion of 3,5-di-tert-butylcatechol [37]. The coordination

Fig. 11. Dependence of the initial reaction rate on the catalyst andsubstrate concentrations for the catalyzed oxidation of 2,6-di-tert-butylphenol, at 25.0�0.5 °C. (A) [2,6-di-tert-butylphenol]=11.5mmol l−1 and (B) [Cu2(apip)2im]3+ =3.50×10−5 mol l−1;[Cu4(apip)4im4]4+ =1.75×10−5 mol l−1.

Fig. 10. Kinetic curves of the catalyzed oxidation of (A) 2,6-di-tert-butylphenol (11.5 mmol l−1), at 25.0�0.5 °C, in methanol solution,monitored at 418 nm. [catalyst]=0.090 mmol l−1, except[Cu4(apip)4(im)4]=0.045 mmol l−1; (B) L-dopa (6.67 mmol l−1), at30.0�0.5 °C, in aqueous solution (phosphate buffer, pH 7.30),monitored at 475 nm. [catalyst]=0.145 mmol l−1.

of the phenolic substrate to the metal centers during thecatalytic cycle, could avoid its subsequent dissociation,after reduction.

4. Conclusions

Our results demonstrated that the dinuclear[Cu2(apip)2im]3+ and tetranuclear [Cu4(apip)4(im)4]4+

complexes are good catalysts for the oxidation of 2,6-di-tert-butylphenol and L-dopa, showing rate constantscomparable to other similar copper species previouslyreported [35,37]. The potentials determined, E1/2=−0.05 V for the mononuclear complex and EPC=−0.07 V for the dinuclear species, agree with the

W.A. Al�es et al. / Inorganica Chimica Acta 321 (2001) 11–21 21

previous studies reporting that the most effective cop-per catalysts in catechol or phenol oxidations showreduction potential close to 0.00 V versus SHE [38]. Inthe presence of the substrate, the catalytically activereduced species are probably kept associated by thecoordinated phenol. The more unstable [SECu(im)-CuSE]+ species, on the contrary, exhibiting only oneNimine coordinated, is probably decomposed after re-duction of the metal centers, which could explain itsinactivity. The better performance of the tetranuclearspecies, showing higher values of k3 and KM than thosefor the corresponding dinuclear complex, could be at-tributed to a more favorable interaction of the phenolicsubstrate in the cavity formed by its cyclic structure, ora more easy release of the product of reaction [36].

Acknowledgements

The support of our research by Fundacao de Am-paro a Pesquisa do Estado de Sao Paulo (FAPESP) isgratefully acknowledged (Grant No. 99/05903-0 toA.M.D.C.F.). W.A.A. also thanks FAPESP for fellow-ship (No. 98/15635-0) during his Ms.Sc. work. Copperanalyses were kindly performed at the laboratory ofProfessor E. de Oliveira. The authors are also indebtedto Dr. J. Mattos for the use of the BOMEM 3.0 IRspectrometer; to Professor H.E. Toma for the use ofelectrochemical apparatus; to M.A. de Oliveira and Dr.M.M. Tavares for the capillary electrophoresis experi-ments, and finally to A.C. Sant’Ana and Dr. M.L.A.Temperini for their assistance with the Raman spectra.

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